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In 1997, we received the first challenging specimen referred from a consultant dermatologist at a hospital belonging to the Ministry of Health in Saudi Arabia for cutaneous leish- maniasis identification by the microscopic smear method. Clin- ically, the lesion looked typical for the dried type of cutaneous leishmaniasis with a painless ulcer and a raised, indurated mar- gin and a necrotic base. On performing the general method for microscopic exam and staining with Wright stain, we were un- ab
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  In 1997, we received the first challenging specimenreferred from a consultant dermatologist at a hospital belonging to the Ministry of Health in Saudi Arabia for cutaneous leish-maniasis identification by the microscopic smear method. Clin-ically, the lesion looked typical for the dried type of cutaneousleishmaniasis with a painless ulcer and a raised, indurated mar-gin and a necrotic base. On performing the general method formicroscopic exam and staining with Wright stain, we were un-able to identify the Leishman-Donovan (LD) bodies within themacrophages, which is the principle of the microscopic diagno-sis of cutaneous leishmaniasis. Afterwards, for the next 10 yearsin practice, we had several similar cases, and we failed todemonstrate the amastigote presence in approximately 50% of the cases by microscopic smear.The purpose of our study was to microscopically identify any extracellular form of Leishmania  present in human lesions and, if found, to describe the new form microscopically and its transfor-mation sequence on a step-by-step basis with photo support. Literature Review  The group of diseases known as the leishmaniasis arecaused by protozoa of the genus Leishmania  . These are presentin 3 different forms: visceral leishmaniasis (VL), muco-cutaneous leishmaniasis (ML), and cutaneous leishmaniasis(CL). The visceral form (kala-azar) is the most severe form of the disease and, if left untreated, is usually fatal. Muco-cutaneous leishmaniasis is caused by Leishmania braziliensis  orrelated New World species, and parasites may disseminate tothe oral and nasopharyngeal mucosa.Cutaneous leishmaniasis is the least severe. It is endemic inover 70 countries. The yearly incidence is estimated at 1.5 mil-lion cases. Over 90% of the cases of CL occur in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Thegeographical distribution of CL is mainly determined by sandfly vectors ( Phlebotomus  sp and Lutzomyia  sp). They live indark, damp places and are relatively weak flyers, with a range of only 50 meters from their breeding site. They are most activein the evening and at night. 1 Leishmania  researchers agree on the following:1) In vertebrates, including human hosts, Leishmania  species are obligate intracellular parasites of mononuclearphagocytes 2,3 ; 2) There are 2 forms of the parasite: the amastigote form, which is intracellular and specifically found in the vertebratehosts including humans, and the promastigote form, predomi-nately found in the insect vector and not found in humans 4 ; and3) The demonstration of the amastigotes and not the pro-mastigote form in lesions plays an important role in thediagnosis of CL. 5,6 Following a sand fly bite, some of the flagellates, once incirculation, enter the cells of the reticuloendothelial system,and there they transform into amastigote forms. The amastigoteforms then multiply by binary fission within the macrophageuntil the host cell is packed with the parasites and ruptures,liberating the amastigotes into circulation. Then the freeamastigotes invade fresh cells, thus repeating the cycle. In theprocess, some of the free amastigotes are drawn by the sandfly during its blood meal, thus completing the cycle. 2 Pathology of the Disease  According to Hepburn, “over the following months, there is a gradual decrease in the number of amastigotes andmacrophages, leaving a granulomatous infiltrate consisting of lymphocytes, epithelioid cells, and multinucleate giant cells. Science 38  LAB MEDICINE  Volume 39 Number 1   January 2008 labmedicine.com Is the Amastigote Form of Leishmania  the Only Form Found in Humans Infected With CutaneousLeishmaniasis? Mohammed Wael Daboul, DDS, MSc, MT(ASCP) (Central Committee of the General Health Laboratories, Ministry of Health, Damascus, Syria) DOI: 10.1309/F0LPBC27FEDQA7RQ  Abstract B    a    ckgro    und: Previous studies of Leishmania  revealed that 2 forms of the parasite arepresent:the intracellular amastigote formfound in the vertebrate host,and thepromastigote form predominately found in theinsect vector. Met    h    o    ds: Samples were collected from thelesion area of the skin of 42 patients referredto the laboratory and microscopic slides wereprepared. R    e    sults:  Out of 42 cases,only 1 (2.4%)showed the presence of the amastigoteintracellularly alone but not in the extracellulararea,and 2 (4.75%) showed the presence ofthe amastigote in the extracellular but not inthe intracellular location.In 20 of 42 cases(48%),we observed the amastigote presencein the intracellular or extracellular area.Out ofall 20 cases in which the amastigote wasrecovered,we were able to identify theamastigote in 17 cases (85%) in theintracellular and extracellular locationssimultanously.Additionally,out of all 42 casesreferred,the amastigote form alone,with nopromastigote associated,was microscopicallyrecovered in 10 cases (24%). C    on    clusi    o    n: Contrary to previous studies,wefound that in the human body Leishmania  doesnot act as an obligatory intracellular parasite. Leishmania  is an intracellular and extracellularparasite infecting the mononuclear phagocytefirst.There,it remains in the form ofamastigote and multiplies by binary fission.When released from the mononuclearphagocyte,after membrane rapture to theextracelluler fluid,the amastigote transformsinto a promastigote-like form in a progressivesequence.At a later stage,this promastigote-like organism continues its development andtransforms again to produce pseudofiber,leaving the lesion area with a permanent scar.   At this stage it may be difficult or even impossible to detectorganisms in H&E, or Giemsa-stained sections.” In the clinicalfeatures, he writes, “most patients have 1 or 2 lesions, usually onexposed sites, varying in size from 0.5 to 3 cm in diameter. Thereis, however, considerable variation: some lesions do not ulcerate,others develop sporotrichoid nodular lymphangitis. Most lesionsheal over months or years, leaving an atrophic scar.” 1  An interesting question arises: why is healing notcomplete without a permanent scar?Referring to Sharquie and colleagues, “the morphology of LD bodies (amastigotes) in smears assumed mainly spindleshape, other morphological forms like barrel, safety pin andumbrella-like were noticed, while the morphology inhistopathological sections were rounded with a nucleus andkinetoplast. However, in some sections spindle shape form simi-lar to smear morphology was detected. LD bodies were seen inhistopathological sections in 30% of patients. Otherhistopathological features were mainly abundant with lympho-cytes and plasma cells in the wet ulcerative lesions while in dry nodular types there was a tendency to form granuloma with lesslymphocytes and scanty plasma cells.” 7 This raises another very interesting question: where did theamastigotes and their macrophages disappear to causing the LDbodies to be seen in histopathological sections in only 30% of patients? And as long as the lesions continue months to a year with no amastigote presence (which means the disappearance of the causing agent of the disease), is it possible that any kind of amastigote transformation has happened and a new form wasmissed by microscopic smear exam? Methods of Transmission The predominant mode of transmission is the bite of a sand fly; however, there are also uncommon modes of transmis-sion through congenital transmission, blood transfusion, and,rarely, through inoculation of cultures. 2 In some regions, nativesinoculate their children in a site normally not visible to protectthe child from developing disfiguring scars later in life. 8  With other modes of transmission aside from the bite of a sand fly raises doubts about the absoluteness of the sand fly vec-tor for the parasite to complete its life cycle, and thus assuming the possibility of the promastigote form to be present in theextracellular fluid of the infected vertebrate. Materials and Methods Since 1999, we have received 42 patients clinically diagnosed with CL referred to us from consultant dermatologists for confir-matory laboratory diagnosis. Our method of identification wasparasite diagnosis by microscopic examination of the skin lesion, which remains the “gold standard” with its usual limitations. Samples Patients referred to the laboratory had samples collectedfrom the lesion area of the skin and microslides were prepared.Two slides were prepared from each lesion. In cases where there were more than 2 exposed areas on the same patient, we chosethe 2 more edematic lesions and took 2 slides from each,stained with Wright stain. All the smears for each case were reserved and numbered assuch: case one (1c), case one slide one (1c1), case 2 (2c), and soforth. Comparison was made among the microscopic features of the parasite in the different slides as follows:1) A study was done for counting the amastigote appear-ance in the intracellular or extracellular location in all cases.2) Another study was done to identify and compare themanifestations of the different shapes of the amastigotes seen in the extracellular fluid among the cases referred.3) Then a study was done for locating the presence of any type of suspicious organism appearing different than any of theblood or the human dermal tissue components.4) A morphologic comparison was made on the discoveredflagellates among the different smears taken from the differentcases referred to us.5) Another morphologic comparison was made betweenthe presumed fiber-forming promastigote form found, and fi-broblasts in skin biopsy specimens stained with H&E stain torule out the difference. Six hundred documentary microscopic photos were takenfor the presumed parasite forms discovered for confirmation, doc-umentation, and comparison with other reference photos for theparasite in both its amastigote and promastigote forms. Results Table 1 illustrates the intracellular or extracellular presenceof the amastigote, and it reveals that out of 42 cases referred tothe laboratory, microscopically, only 1 case (2.4%) showed thepresence of the amastigote intracellularely alone but not in theextracellular area; 2 cases (4.75%) showed the presence of theamastigote in the extracellular but not in the intracellular loca-tion; and in 20 cases (48%), we microscopically observed theamastigote presence in the intracellular or extracellular area.Out of the 20 cases in which the amastigote was recovered, we were able to identify the amastigote in 17 cases (85%) in theintracellular and extracellular locations simultanously. Addi-tionally, in all 42 cases referred, the amastigote form alone— with no promastigote associated—was microscopically recovered in 10 cases (24%). Table 2 shows other forms found in the microscopic slidesof the 42 patients and reveals that out of 42 cases referred tothe laboratory, microscopically, the promastigote-like form wasfound in 32 (76%) of the cases referred, and the amastigoteform was recovered associated with the promastigote-like in 10 Science labmedicine.com January 2008  Volume 39 Number 1   LAB MEDICINE  39 Table 1 _Presence of the Amastigote Form  Amastigote FormIntracellular Alone Extracellular AloneOnly Amastigote Present Amastigote Present Inside or With No Promastigote FormOutside the Macrophage No.of cases1/422/4210/4220/42Percentage2.384.752448  cases (24%) of the total. The promastigote form was recoveredalone without the amastigote combination in 22 cases (52%). Discussion Table 1 indicates that the amastigote forms are almost avail-able in both intracellular and extracelluler fluid simultaneously every time the amastigote form is found in the smear. That pres-ence in the extracelluler fluid demonstrates the amastigote’s abil-ity to survive in the extracelluler environment not necessarily thesame as inside the macrophage, but at least as a bridge for trans-formation into the promastigote-like form. This leads to theconclusion that, in humans, the amastigote form (contrary tothe previous studies) is not an obligatory intracellular parasitethat cannot survive in the extracelluler environment as long as we see it in both locations at almost the same rate.From Table 2  we concluded that the images ( Images 1–12 )have proven the presence of other forms apart from the amastig-ote form in the extracelluler fluid. We called these forms “pro-mastigote-like” as a general term, and, according to theirdevelopmental stage, and relying on the photos taken from theslides, we classified them in the following order:1) Once outside the macrophage, the amastigote form be-comes like an ova containing the promastigote embryo. The nu-cleus is usually centralized, the surrounding cytoplasm has a spindle-like shape, and the chromatin is smooth ( Images 1,2 ,and 5 ).2) Inside the amastigote, the nucleus starts to take a polariz-ing position, and the cytoplasm appears on one side of theamastigote. At the same time, the chromatine shows more con-densation ( Images 1 and 2 ).3) This developing embryo becomes larger in size and morecondensed, assuming a candle flame shape while the cytoplasmdisappears ( Images 1 and 8 ).4) The embryo continues to grow with a small tail protrud-ing outside ( Images 1,7 and 8 ).5) The tail continues growing, taking a flagella shape, to-gether with the whole organism increasing in size from 2 to 3microns to approximately 4 to 6 microns, and starting to assumea small promastigote shape ( Images 7 and 8 ).6) In this stage, the promastigote becomes fully mature with dimensions approximately 8 to12 microns in diameter ( Images 3–6 and 8 ).Sharquie and colleagues wrote, “the morphology of LDbodies (amastigotes) in smears was mainly spindle in shape,other morphological forms like barrel, safety pin and umbrella like were noticed.” 7 In fact, they described the transforming stages of the amastigote to promastigote-like but were not ableto identify the transformation significantly. 7) Transformation into fiber-forming promastigote stage. In this stage the promastigote-like form starts transforming intofiber production where the flagella becomes more condensed,thickened, and enlarged to approximately 40 microns or morein length ( Image 10 ). We may also see the fiber formation notonly from the flagella position but also from the opposite side( Image 12 ), or occasionally at different poles ( Image 11 ). Thisfiber structure formation in the derma may react like a foreigngraft and explain the inflammatory fiber granulomatous immunereaction associated with the lymphocytes, mononuclear cells, andvariable number of plasma cells (graft rejection-like reaction).Once again, Sharquie and colleagues described that “otherhistopathological features were mainly abundant of lymphocytesand plasma cells in the wet ulcerative lesions, while in dry nodu-lar types there was a tendency to form granuloma with less lym-phocytes and scanty plasma cells.” 7 They were able to describethe histopathological condition of the disease without connect-ing it with the etiology, which is the presence of the promastig-ote in the wet lesion and the presence of fibers and fiber-forming parasites in the dry nodular type of lesions. Our findings indi-cated we could see a mixture of both histopathological featuresmentioned at different rates, in different smears according to a certain stage of the disease case. 8) The last phase: conversion to fibers. Here the fibers areloaded, elongated, and the parasite nucleus becomes thinner,smaller, thready, and more condensed in the middle, along withthe fiber continuing from both sides ( Image 9-12 ). It is as if theparasite embalms itself. At this point, few lymphocytes andplasma cells are seen and, clinically, the lesion is dry and close tohealing and forming a permanent scar.The formation of the post-healing lifetime scar is justifiedby the production of these types of pseudo-fibers in the lesion by the parasite itself and not the human fibroblasts. Apparently,those remaining foreign fibers formed and inserted in the laststage of the lesion healing will stimulate and become a focal area of permanent immune inflammatory reaction, and this explainsin part the incomplete healing of the skin in the area with result-ing permanent scar formation.Some researchers went through this phenomena. Hepburn wrote, “there is, however, considerable variation: some lesions donot ulcerate, others develop sporotrichoid nodular lymphangitis.Most lesions heal over months or years, leaving an atrophicscar,” 1 but without detecting the reason of this lifetime scar.  L M  Acknowledgment:  I indeed thank Dr. Tayseer Daboul, Dr.Robert Singletary, Dr. Rafael A. Cuello, and Patricia A. Clark,MS, MT(ASCP), for their efforts, encouragement, support, andconsideration. 1.Hepburn NC. Cutaneous leishmaniasis: An overview.  J Postgrad Med. 2003;9:50–54. 2.Vidyashankar C, Agrawal R. Leishmaniasis  . E-Medicine Specialties. Availableat: www.emedicine.com/ped/topic1292.htm. Last Updated: February 27,2006.3.Cascio A, Calattini S, Colomba C, et al. Leishmaniasis. Polymerase chainreaction in the diagnosis and prognosis of Mediterranean visceral leishmaniasisin immunocompetent children. Pediatrics  . 2006;109:e27–27 .4.Parasitism & Symbiosis. 177-345A. Leishmania  . McGill University.Department of Biology.5.Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Kala-AzarMedical Research Center, Department of Medicine, Banaras Hindu University,Institute of Medical Sciences, Varanasi 221 005, India.6.Beena KR, Ramesh V, Mukherjee A. Identification of parasite antigen,correlation of parasite density and inflammation in skin lesions of post kala-azar dermal leishmaniasis.  J Cutan Pathol. 2003;30:616–620.7.Sharquie KE, Hassen AS, Hassan SA, et al. Evaluation of diagnosis of cutaneous leishmaniasis by direct smear, culture and histopathology. Saudi  Med J. 2002;23:925-928.8.World Health Organization. Leishmaniasis  . Disease information. TDR diseases. Available at: www.who.int/tdr/diseases/leish. Science 40  LAB MEDICINE  Volume 39 Number 1   January 2008 labmedicine.com Table 2 _Presence of Promastigote-Like Forms Promastigote-LikeSeen in Associated With thePresent Alone in Formsthe Smear Amastigote Formsthe Smear (With no Amastigote Forms) No.of cases found32/4210/4222/42Percentage762452  Science labmedicine.com January 2008  Volume 39 Number 1   LAB MEDICINE  41 Image 1Image 2Image 3Image 4Image 5Image 6Image 7Image 8Image 9Image 10Image 11Image 12
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